Summary Our overall project goal is to systematically link c/s-regulatory elements in pre-mRNAs to RNA structural features that control alternative pre-mRNA splicing in human cells. While the role of pre-mRNA structure in control of splicing events has been studied for individual genes, a comprehensive understanding of the interplay of RNA structure with splicing control?and its relation to genetic variation?is not known for the human transcriptome. Our aims are to: 1) Map nuclear pre-mRNA structure in vivo and in vitro using high-throughput chemical probing methods and test the effects of RNA chaperone proteins on those structures. 2) Determine the positioning of mapped pre-mRNA structures relative to splicing factor binding sites, splice sites and exons, and human genetic variants. 3) Define a role for RNA chaperone proteins in alternative splicing decisions in human cells. 4) Develop and validate RNA maps relating RNA chaperone binding, RNA structure, and pre-mRNA splicing events, and validate roles of predicted structures in alternative splicing. These models will incorporate biochemical data, splicing reporter assays, as well as phylogenetic information and human genetic variation. Our efforts will initially focus on the genome-wide mapping of single-stranded RNA regions detected by chemical probing of pre-mRNA structure, as influenced by two known RNA chaperone proteins, the hnRNP protein hnRNPA1 and the p68/DDX5 RNA helicase. The RNA binding and chaperone activities (RNA annealing or unwinding) of hnRNP A1 and p68/DDX5 helicase will be correlated to global RNA structural data and alternative splicing patterns. We want to define instances where RNA structure plays a role in alternative splicing decisions and then integrate genome-wide data sets into these models. Our ultimate goal will be to generate systems level network models for how RNA structure and RNA chaperones, human genetic variation and histone marks, and RNA polymerase II distribution control alternative pre-mRNA splicing to inform both mechanistic aspects and the underlying molecular basis of human disease gene mutations.